The Leishmania spp. protozoa have a profound effect on the host cell that they invade. These parasites reside intracellularly usually in macrophages, a cell type with the capacity to kill intracellular microbes. Rather than succumb to microbicidal mechanisms, Leishmania paralyze the microbicidal pathways of the host cell, changing the macrophage from a lethal cell to an intracellular safe haven which allows the parasite to survive, replicate and ultimately spread to neighboring cells. The mechanism(s) through which the parasite "paralyzes" the host microbicidal function is incompletely understood. It has recently come to light that most eukaryotic organisms utilize short noncoding RNA sequences to globally regulate expression of a wide variety of genes. Indeed short RNA sequences of 18-30 bp in length, called microRNAs, are critical for regulating expression of an estimated 30% of the human genome. Although microRNAs can trigger mRNA degradation, many changes induced by microRNAs affect gene expression at the level of translation. Thus the effects of microRNAs may be evident through studies of protein, but not necessarily mRNA abundance. The hypothesis underlying this proposal is that microRNAs are the upstream trigger(s) responsible for the global changes in macrophage gene expression induced by phagocytosis of Leishmania chagasi. Furthermore, we hypothesize that leishmania manipulate these microRNAs as a means of converting the macrophage in to an environment compatible with its own survival. The specific aims of this proposal are: 1. To perform a global profiling of changes in microRNA expression induced during and after phagocytosis of L. chagasi by human macrophages. 2. To correlate changes in microRNAs with potential targets in 3'UTRs, and with changes in protein expression using a human antibody microarray. 3. To examine in vivo in a mouse model whether correlated changes occur in the livers of infected mice, using microRNA microarray chips that contain murine sequences. Immediate future goals of this 2-year R21 project will include experimental introduction or knock-down of microRNAs in macrophage cultures and in vivo in an effort to systematically test the biological effects of microRNAs identified herein. Ultimate goals include the extension to a human system, with the intent of developing novel therapeutic tools. PUBLIC HEALTH RELEVANCE: RELEVANCE TO PUBLIC HEALTH Latin American visceral leishmaniasis is a potentially fatal human disease caused by the protozoan Leishmania chagasi. During this study we will systematically investigate the role of microRNAs, which are small regulatory molecules, in the progression of Leishmania chagasi infection. Future goals of this research include the use of microRNAs to develop novel targets for treatment of human leishmaniasis.